CN113924001A - Food supplement - Google Patents

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CN113924001A
CN113924001A CN202080016194.8A CN202080016194A CN113924001A CN 113924001 A CN113924001 A CN 113924001A CN 202080016194 A CN202080016194 A CN 202080016194A CN 113924001 A CN113924001 A CN 113924001A
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supplement
galactose
food supplement
group
composition
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雷米·普拉德莱斯
安托万·德尔布吕特
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MICROPHYT
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A23L33/11—Plant sterols or derivatives thereof, e.g. phytosterols
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system

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Abstract

The present invention relates to a composition comprising at least 50mg/g of one or more omega-3 type fatty acids, at least 10mg/g of one or more xanthophylls, at least 1mg/g of one or more sterols and at least 2 μ g/g of one or more algal prostaglandins, and its use, in particular as a food supplement, for preventing the appearance of cognitive disorders.

Description

Food supplement
Technical Field
The present invention relates to a composition, as well as a fatty acid and lutein based food supplement and its use, in particular for preventing cognitive disorders in humans or animals.
Background
Cognitive processes are defined as all brain functions that allow the acquisition, processing, memory and use of data from the environment in order to maximize the advantages and reduce the drawbacks of external constraints. Thus, the cognitive process is implemented at stages involving reasoning (generating plans, organizations, judgments), perception, recognition, language, emotion, memory, and learning.
Mild cognitive impairment or cognitive vulnerability is defined as no change in cognitive function with dementia. From a clinical point of view, these disorders are associated with a score of 0.5 in the case of evaluation by CDR (cognitive drug research computer evaluation system) test.
Of these cognitive disorders, age-related cognitive decline and prenatal stress-induced cognitive changes are two phenomena that may affect one's lifetime.
Cognitive decline associated with aging is defined as a non-pathological decline in cognitive function, such as speed of information processing, attention capacity, and in particular so-called working (or short-term) memory. These processes are caused by normal physiological changes directly associated with aging. The age at which this decline begins is still controversial, but considering the accelerated aging of the world population, over 20% in the world over 60 years old, and over 30% by 2050, the age-related cognitive decline is one of the major problems in the next decades, and it has a major impact on economy (reduced autonomy for the elderly) and public policy worldwide, particularly in developed countries.
In contrast to the age structure pyramid, cognitive impairment may affect infants and young children under prenatal stress. Indeed, the effect of stress during certain periods of pregnancy on cognitive development in unborn individuals has been studied in humans and animals for several years. Thus, in animals, mainly in rats, maternal prenatal stress has been shown to result in alterations in long-term memory of offspring.
It has been demonstrated that strong negative stimuli, stress, may lead to non-pathological alterations or a decline of cognitive function in young children, manifested as hyperactivity, deficits in attention and memory, language development retardation, more difficult-to-meet splenic qi and more general behavioral alterations such as anxiety behavior, showing a delay in neurodedevelopment and a decline in cognitive ability.
One of the proposed mechanisms for expressing prenatal stress as a cognitive disorder is based on exposure of the fetus to large doses of so-called stress hormones, which belong to the family of corticosteroids, such as cortisol. However, cortisol crosses the placental barrier and, starting from a certain concentration, the protection mechanisms of the fetus from the corticoids secreted by the mother are saturated, exposing the fetus to excessive doses of cortisol, which seems to have a negative impact on cognitive development. Other complementary hypotheses have been developed to explain the relationship between prenatal stress and cognitive impairment in children.
The concept of pressure can be defined according to different angles, such as biological methods: stress is a series of metabolic reactions that, after one or several exogenous factors, cause physiological or psychological changes (fear, distress) in the organism. However, the concept of stress and its impact is largely personal, and the individual's response to stress is also defined from a psychological point of view. Since then, remembering that is a retrospective stress, because everyone has a specific response, or because it may be an objective stress, it is difficult to take action on the source of the prenatal stress. In addition, pregnancy can cause hormonal and psychological changes that increase the susceptibility of the expectant mother to everything that may affect the health of the unborn baby.
Strict treatment of stress or anxiety in the form of a pharmaceutical prescription is dangerous for pregnant women: many psychotropic drugs for the treatment of psychological disorders or anxiety have teratogenic effects with direct adverse effects on the fetus. This requires evaluation on a case-by-case basis and is only used in the case of clinical psychological disorders of the pregnant woman and not in the case of so-called subjective stress.
Thus, there is a major problem in finding a solution to the consequences of prenatal stress on cognitive impairment in children or young adults.
Different studies have shown that nutritional supplementation of so-called essential fatty acids and carotenes, in particular lutein, and even combinations of the above mentioned fatty acids and carotenes, is of interest for preventing or at least limiting the decline of cognitive function. Food supplements or drugs have been developed and positive results have been achieved.
Thus, according to the document WO2013/032333a1, a composition based on omega-3 type fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), astaxanthin and glycerophospholipids is known, suggested for the prevention or treatment of various diseases, in particular cognitive disorders. Said ingredient is present in the composition in the form of a microalgae extract; in a preferred preparation variant, they are obtained by formulating two extracts derived from two different algae. The natural origin of the ingredients of the composition is a great advantage. Nevertheless, a need still exists for more effective compositions, particularly with respect to the above-mentioned problems. Furthermore, it is important to provide a simple and reproducible method of preparing such compositions.
Disclosure of Invention
The present invention provides a regimen of a composition comprising one or more omega-3 type fatty acids and one or more xanthophylls, as well as one or more compounds of the sterol family and one or more algal prostaglandins. It has been found that the combination of at least one sterol and at least one algal prost with at least one omega-3 type fatty acid and at least one lutein greatly increases the effectiveness of the composition in preventing the appearance of age-related cognitive impairment and also prevents disorders related to prenatal stress.
The composition of the present invention comprises:
at least 50mg/g of one or more omega-3 type fatty acids,
at least 10mg/g of one or more xanthophylls,
at least 1mg/g of one or more sterols, and
at least 2 μ g/g of one or more algal prostaglandins.
In the main indications, the composition of the invention can be used as a food supplement. Furthermore, the present invention relates to a food supplement comprising at least 50mg/g of one or more omega-3 type fatty acids, at least 10mg/g of one or more xanthophylls, at least 1mg/g of one or more sterols, and at least 2 μ g/g of one or more algal prostaglandins.
The main advantage of the present invention is that all the above components or constituents can be obtained from natural sources, in particular they can be extracted from one or more microalgae, preferably from one single microalgae. Of course, the components or ingredients of the composition or food supplement of the present invention may be of non-natural origin and provided in the form of a chemically synthesized product.
Before disclosing the present invention in more detail, some of the terms used herein are defined.
In the expression "composition comprises" or "food supplement comprises" the word "comprising" means that the composition or supplement may comprise any additional ingredient or ingredients not specifically mentioned, in any form and from any source. It also includes compositions or supplements containing only the listed ingredients, and thus consists of the above ingredients.
A food supplement is defined as one or more food products whose purpose is to supplement the normal diet of a human or animal and constitute a concentrated source of nutrients or other substances with a nutritional or physiological effect, alone or in combination; it is usually provided in dosage form, i.e. in packaged form, such as gel capsules, lozenges, tablets, troches and other similar forms, as well as in powder packs, liquid ampoules, dropper-containing vials and other similar forms of liquid or powder preparations intended to be taken in small metered units.
omega-3 fatty acids are a class of unsaturated fatty acids having a hydrocarbon chain of 4 to 36 carbon atoms, typically 14 to 36 carbon atoms, and a double or first double bond on the third carbon-carbon bond, as measured from the terminal methyl group of the chain. The unsaturation may be cis or trans, independently of each other. The most representative acids are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but the name "omega-3 type fatty acids" is not limited thereto. Furthermore, especially when the fatty acids are derived from natural sources, they may be extracted from algae and present in the form of free molecules, but also in the form of derivatives, for example esterified forms, such as mono-, di-or tri-esterified forms, or mixtures of these forms.
Xanthophyll is defined as a molecule belonging to carotenoids and comprises one or several oxygen atoms, such as astaxanthin, canthaxanthin, adiabatidine, lutein, zeaxanthin, diadinoxanthin, neoxanthin, chlorethaxanthin, siphonaxanthin, diatoxanthin, violaxanthin, chrysophanthin, alpha-cryptoxanthin, beta-cryptoxanthin and fucoxanthin. In particular, when the xanthophylls are derived from natural sources, they may be extracted from the seaweed and present in the form of free molecules, or may be present in the form of derivatives, such as esterified forms of monoesters or polyesters, or mixtures of these forms.
Sterols are a well-known class of lipids having a stanol core with a hydroxyl group at the 3-position, which can be modified, for example, with acetyl groups. These include natural sterols or phytosterols and are included herein by the term phytosterol. Without being limited thereto, as examples of phytosterols, there may be mentioned 24-methylene cholesterol, β -sitosterol, fucosterol, isofucosterol, sargasterol, oxocholesterol acetate, lilium maritime sterol, more particularly brassicasterol, stigmasterol, and campesterol.
With algal prostaglandins, it is understood that the lipids structurally belong to the prostaglandin class of lipids, from natural sources, produced by the indirect enzymatic oxidation of fatty acids naturally present in microalgal biomass. In particular, these compounds are selected from the group consisting of phytoprost, isoprostane and neuroprostane, depending on the fatty acids which have undergone oxidation. Thus, these compounds may be derived from fatty acids, such as alpha-linolenic acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). The plant prost is mainly derived from ALA, and can be selected from 9-epi-9F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, 9-F1t-PhytoP, ent-16B1t-PhytoP, ent-9L1t-PhytoP, and 16(RS) -16-A1 t-PhytoP. The isoprostane is mainly derived from ARA and EPA, and can be selected from 15-E2t-IsoP, 15-F2t-IsoP, 15-epi-15-F2t-IsoP, 5-F2t-IsoP, and 8(RS) -8-F3 t-IsoP. The neuroprostaglandin is mainly derived from DHA and can be selected from 4-F3t-NeuroP, 10-F4t-NeuroP, 10-epi-10-F4t-NeuroP, 4(RS) -4-F4t-NeuroP, 14(RS) -14-F4t-NeuroP, and 20(R) -20-F4 t-NeuroP.
Medium Chain Triglycerides (MCT) are understood to be esters of glycerol and saturated fatty acids, the hydrocarbon chain of which has from 6 to 12 carbon atoms. They are naturally found in coconut oil, palm kernel oil, palm oil and the like, but can also be obtained from other fats and oils.
The invention is described in more detail below and variants thereof are disclosed.
Advantageously, the composition or food supplement of the invention presents the following characteristics, considered alone or in any combination.
It comprises 50-250mg/g of one or more omega-3 type fatty acids, 10-50mg/g of one or more xanthophylls, 1-20mg/g of one or more sterols, and 2-100 μ g/g of one or more algal prostaglandins.
It comprises 50-200mg/g of one or more omega-3 type fatty acids, 10-30mg/g of one or more xanthophylls, 1-8mg/g of one or more sterols, and 2-50 μ g/g of one or more algal prostaglandins.
It comprises 50-170mg/g of one or more omega-3 type fatty acids, 10-25mg/g of one or more xanthophylls, 1-6mg/g of one or more sterols, and 2-40 μ g/g of one or more algal prostaglandins.
Advantageously, the composition or food supplement of the invention further comprises at least one oil as a carrier or support to promote the expression of the active ingredient. Surprisingly, it has been observed that when the oil is selected from Medium Chain Triglycerides (MCT), the production of the composition or food supplement is facilitated. In particular, when the active ingredients are obtained from the same microalgae extract, optimal homogenization is observed in this oil. According to one variant, the Medium Chain Triglycerides (MCT) are derived from natural sources, brought about by oils selected from coconut oil, palm kernel oil and palm oil; they may also be obtained or derived from such oils.
In the following, preferred formulations of the composition or food supplement of the invention are presented, these implementations can of course be combined:
the omega-3 type fatty acids or at least one omega-3 type fatty acid is selected from stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and mixtures thereof;
the or at least one xanthophyll is fucoxanthin;
the or at least one sterol is selected from phytosterols;
the algal prost or at least one algal prost is selected from the group consisting of a plant prost, an isoprost, and a neural prost.
The composition or food supplement of the invention may comprise any additive that may improve, inter alia, its preservation, appearance, taste, formulation. Thus, one or more additives, such as those selected from preservatives, coloring agents, flavoring agents, disintegrating agents, lubricating agents, coating agents or encapsulating agents, may be added thereto.
The main application of the composition of the invention is in nutrition, and therefore such composition or food supplement as defined above is advantageously in the form of gel capsules, tablets, lozenges or loose powders. Preferably, it is packaged in doses having a unit weight between 1mg and 1 g. In general, the galenical formulation of the composition or supplement will be adapted to the individual in question, in particular depending on whether it is for children or adults.
The composition or food supplement of the invention can be used to prevent the appearance of non-pathological aging-related cognitive disorders or non-pathological cognitive disorders in children or young adults suffering from prenatal stress. In the prevention of age-related cognitive disorders, daily intake may be comprised between 2 and 5mg/kg body weight. In the prevention of cognitive impairment in children or young adults suffering from prenatal stress, daily intake may be comprised between 0.05 and 0.1mg/kg body weight. It has been noted that as long as the duration of the treatment is proportionally prolonged, effects can be observed even for very small daily intakes.
The invention also relates to the use of microalgae for preparing a food supplement as defined previously. One or more preferred microalgae are selected from any of the following taxonomic groups: lipoidyceae, Chryseophyceae, Diatomae, Phaseolus, Prymophyceae, Didinoflagellaceae, Coccolithophyceae, Isodinoflagellaceae, and Phaeodaceae. Advantageously, the microalgae are dinoflagellates luteinii or phaeodactylum tricornutum. Such microalgae are chosen because appropriate extraction results in the components of the extract meeting the definition of the composition of the invention. For example, such an extract may comprise the following fatty acid moieties: fatty acids, expressed in percentages by weight with respect to the total extract, are comprised between 4 and 55% in the form of free fatty acids, between 0.5 and 10% in the form of monoacylglycerols, between 0.4 and 15% in the form of diacylglycerols, and between 2 and 55% in the form of triacylglycerols. These fatty acids are omega-3 series fatty acids in the range of 5-20% (m/m) and omega-6 series fatty acids in the range of 0.5-5%. More particularly, in particular, the fatty acids are 0.5-10% ALA (alpha-linolenic acid), 0.5-10% SDA (stearic acid), 0.05-20% EPA (eicosapentaenoic acid), 0.1-10% DHA (docosahexaenoic acid).
As mentioned previously, one of the benefits of the composition or food supplement lies in the method of preparation thereof, in particular the natural source of its ingredients, all of which are obtainable from one single microalga. Depending on the microalgae used, the preparation of the composition may be obtained directly from the extract. If this is not the case, the extract will be diluted to obtain the desired concentration according to the invention. However, the invention is not so limited and thus, it is contemplated that only a portion of the components are derived from natural sources, and others are obtained by chemical synthesis and/or are not derived from the same source, e.g., they are not produced from the same algae.
Measuring and adjusting the concentration of the active ingredient in the extract, composition or obtained food supplement can be carried out using analytical techniques belonging to the general knowledge of a person skilled in the art.
Methods of producing compositions or food supplements from microalgae cultures are described in more detail below.
According to a variant of the invention, the organism is a microalgae, for example an organism belonging to the taxonomic group lipochaeta, chrysophyceae, diatoms, phaeophyceae, prohnophyceae, dinoflagellates, Coccolithophyceae, Isodinoflagellates, Phaeodactylaceae, etc. These photosynthetic microorganisms may be strictly autotrophic, mixotrophic or temporarily heterotrophic. These organisms can be harvested in the natural environment or preferably in a culture environment.
Extract means the fraction of biomass derived from an organism having photosynthetic capacity, obtained by a process allowing to obtain directly or indirectly the composition of the invention. These extracts have a composition expressed in weight percentages with respect to the total extract, comprising between 5% and 30% of proteins, between 20% and 80% of lipids, between 0.1% and 2% of sterols, between 0.1% and 20% of chlorophylls.
More specifically, the lipophilic part constituting the extract consists, expressed in percentage by weight with respect to the total extract, of 15-45% of saturated fatty acids, 5-20% of polyunsaturated acids, 1-20% of lutein and 0.0002-0.007% of algal prost.
For the production of the extract according to the invention, the cells advantageously consist of microalgal cells of the species dinoflagellate luteum of the family isodinoflagellaceae or of the species phaeodactylum tricornutum of the family phaeodactylaceae produced by carbon autotrophy.
Production method of microalgae
It is desirable to culture microalgae in a controlled manner in a suitable system, such as a pipeline, an open pond, or preferably in a closed system such as a photobioreactor. The photobioreactor used may be of any existing type, such as a horizontal tubular photobioreactor, a vertical, e.g. so-called "green wallboard" system, a planar or a cylindrical photobioreactor. Preferably, the production of biomass will be carried out within a closed cultivation system by zero influence autotrophy on arable land.
The production of biomass is carried out according to batch, fed-batch, continuous, semi-continuous, turbidimetric or chemostatic culture management practices.
Obtaining extracts of these microalgae
Extracts derived from these microorganisms are preferably obtained after concentrating the biomass by eliminating all or part of the water content in a drying step using chemical or physical processes, such as centrifugation, filtration, flocculation, precipitation (whether coupled or not), by freeze-drying, vacuum-drying, drum-drying, atomization or any other process that allows to reduce the water content of the biomass. In addition to these steps, cell lysis processes may be performed, such as applying pressure, electrical current, shear forces, using enzymes, or any other process that can destroy a tissue, organ, cell, or organelle.
The target compounds in the biomass are extracted according to a solid-liquid extraction method, which may use a high critical fluid or a subcritical fluid, which may involve co-treatment, e.g. microwave, ultrasound, pressure, enzymes, performed in parallel or sequentially. The solvent used, whether pure or a mixture, may comprise acetone, n-hexane, ethyl acetate, methyltetrahydrofuran, heptane, methanol, natural or branched oils, ethanol or any other solvent from which all or part of the hydrophobic and amphiphilic compounds may be extracted.
The solvent or solvent mixture is separated from the residual biomass after extraction by centrifugation, filtration, etc., and may then be concentrated by vacuum evaporation or any other technique that allows selective evaporation of the solvent in question, or elimination of the solvent. The extract thus obtained is lipophilic and at the same time contains amphiphilic molecules.
Formulation as a food supplement
The formulation of the extract is carried out with a compatible base, making it soluble to obtain a homogeneous solution of the extract with the desired concentration, for example a vegetable oil, such as olive oil, rapeseed oil, linseed oil, sunflower oil, grapeseed oil, palm oil, and preferably MCT oil, and consisting of a mixture of caprylic and capric acids of about 70% by weight, preferably chosen from coconut oil or palm oil, overall supplemented with molecules that allow an increase in stability, such as synthetic or natural antioxidants. To obtain the supplement, the matrix/additive may be added in a weight ratio of up to 95% by weight of the food supplement, they being generally comprised between 15% and 80%, preferably between 35% and 45%.
The extract, but preferably the formulated composition or the obtained supplement, can be formulated in the form of soft capsules by any technique capable of microencapsulating aqueous solutions, and also in the form of powders, which comprise (or not comprise) a support or matrix that allows (or does not allow) its homogeneous dispersion in a drinkable polar solution.
The extract or supplement can be used alone or as an ingredient in a food supplement.
Drawings
The different objects of the invention are explained below with reference to the following figures and their advantages are illustrated in the following examples:
figure 1 is a representation of the effect of the supplement of the invention on voluntary activity, the left panel illustrating the effect on spontaneous alternation disorder and the right panel illustrating the effect on voluntary activity.
FIG. 2 is a representation of the effect of MWM testing on learning impairment caused by D-Gal.
FIG. 3 is a representation of the effect of supplementation and DHA on learning impairment caused by D-galactose.
FIG. 4 shows the effect of D-galactose on passive avoidance in mice, the effect of the diving platform latency is shown in the left panel, and the effect of the escape latency is shown in the right panel, measured during the retention period.
FIG. 5 is a representation of the effect of supplementation and DHA on D-galactose induced lipid peroxidation.
FIG. 6 is a representation of the effect of supplementation and DHA on D-galactose induced TNF- α expression in the cortex and plasma, the effect on the cortex being shown on the left and the effect on the plasma on the right.
FIG. 7 is a representation of the effect of supplementation and DHA on D-galactose induced expression of IL-6 in the cortex (left panel) and plasma (right panel).
Figure 8 is a representation of the effect of supplementation on anxiety in a motor test at the center of the test space (day PPD 46).
FIG. 9 is a representation of the effect of supplements on recognition memory in a test to identify an object (day PPD 47).
FIG. 10 is a representation of the effect of supplements on recognition memory in a test to identify new objects.
Figure 11 is a representation of the effect of the supplement of the present invention on autonomic activity, the left panel illustrating the effect on spontaneous alternation disorder and the right panel illustrating the effect on autonomic activity.
FIG. 12 is a representation of the effect of MWM testing on learning impairment caused by D-Gal.
FIG. 13 is a representation of the effect of supplements on learning disorders caused by D-galactose.
FIG. 14 is a graph showing the effect of D-galactose on passive avoidance disorder in mice, wherein the effect of the diving platform latency is shown in the left panel and the effect of the escape latency is shown in the right panel, measured during the retention period.
FIG. 15 is a representation of the effect of supplements on D-galactose induced lipid peroxidation.
FIG. 16 is a representation of the effect of supplementation on D-galactose induced TNF- α expression in the cortex and plasma, with the effect on the cortex on the left and the effect on the plasma on the right.
FIG. 17 is a representation of the effect of supplementation on D-galactose induced expression of IL-6 in cortex (left panel) and plasma (right panel).
Example 1 formulation of extracts containing Components of the composition of the invention
An extract is obtained from the microalgae phaeodactylum tricornutum according to any of the above techniques.
It is insoluble in water and highly viscous and cannot be handled at room temperature.
The extract and palm oil were kept at room temperature (25. + -. 1 ℃) for 24h before preparation. The extract was transferred to a centrifuge tube containing oil such that the final net mass of the mixture was about 5g and the mass proportion was 25% of the total net mass of the mixture. The mixture is stirred into one unit using a so-called vortex mixing device. Stirring was repeated three times for each mixing. A homogeneous mixture was obtained.
Example 2: natural extracts of the microalgae Isochrysis lutea in an in vivo model attenuate age-related cognition Testing of disorders caused by regression
The food supplement of the invention is prepared from extracts of dinoflagellates, such as luteum, comprising (mg/g):
omega-3 type fatty acids (ALA, SDA, EPA, DHA): 152.6 +/-14.4;
fucoxanthin: 20.0 plus or minus 4.0;
sterol: 4.9 plus or minus 0.8;
algae prostacyclin: 0.035 + -0.007.
The supplement is obtained by adding 360mg + -10 mg/g of coconut oil to the extract.
The supplement was added to the kibbles according to 3 different recipes, with final concentrations of DHA in the kibbles of these batches equal to 0.5, 1.5, and 3.0% (m: m).
A commercial microalgal oily extract containing only the fatty acids DHA 77% (m: m) and EPA 3% (m: m) as the fat fraction was also tested; it was also added to the batch of kibbles, making the final concentration of DHA in the batch equal to 3.1% (m: m).
Another batch of kibbles was formulated with only coconut oil, so that the carrier concentration was equal to the other batches, i.e., 0.01% (m: m).
The five batches of kibble thus obtained are described in table 1 below.
[ Table 1]
Formulated kibble Marking [DHA]％(m:m)
Coconut oil A1 0
Supplement agent A2 0.5
Supplement agent A3 1.5
Supplement agent A4 3.0
Commercial oily extract A5 3.1
The in vivo model considered is a D-galactose model applied to mice, which is suitable for studying cognitive decline associated with aging. In fact, this model mimics many of the behavioral and molecular characteristics of brain aging in rodent models.
D-galactose was administered subcutaneously at a rate of 150mg/kg mouse wet weight per day and the food supplement described herein was incorporated into the pellets according to the following mode:
between day-14 and day 51, the supplement is administered by incorporation into a food pill.
-subcutaneously administering D-galactose between day 01 and day 51, 5 days per week.
Between day 43 and day 51, three different behavioral tests were used to monitor the effect of the test compounds.
The effectiveness of the supplement was evaluated according to the following parameters: improvement of learning disabilities (spatial working memory: spontaneous alternation according to the Y maze test; spatial memory by the so-called "Morris water maze" and long-term situational memory by passive avoidance test), Lipid Peroxidation (LPO) rate of hippocampus and effect on neuroinflammatory markers IL6 and TNF α.
Amelioration of learning disabilities
On day 43, all animals tested spontaneous alternation in the Y Maze (YM) test by spatial working memory index.
All animals were tested for spatial memory in the Morris Water Maze (MWM) test by spatial memory index from day 44 to day 49.
All animals passed the MWM test to assess spatial working memory from day 44 to day 49.
-assessing the long-term situational memory of the animal with a stepwise passive avoidance procedure (STPA) on days 50 and 51, respectively, by exercise and retention sessions.
On days 50 and 51, all animals were tested for the STPA task.
Lipid Peroxidation (LPO) rate of hippocampus and effects on neuroinflammatory markers IL6 and TNF α
On day 51, animals were euthanized at the end of the behavioral testing.
For all animals, trunk blood was collected and centrifuged to recover plasma, and brains were collected rapidly. Dissecting hippocampus and cortex, and determining lipid peroxidation rate by colorimetry using hippocampus; the prefrontal cortex and plasma are used to determine the levels of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha).
Quantification of Lipid Peroxidation (LPO) rates was performed according to the Hermes-Lima et al modified and adapted procedure. The method measures the ability of brain lipid peroxides to oxidize the ferrous oxide and xylenol orange complex, performed in the presence of cumene Hydroperoxide (HPC). Lipid peroxidation levels were determined as HPC equivalents according to the following formula:
HPCE＝A5801/A5802 x[HPC(nmol)]。
and expressed as HPC equivalents per wet tissue weight and as a percentage of the data obtained with the control (D-galactose + vehicle).
The levels of IL6 and TNF α were quantified by ELISA assay using the following kit:
for quantification of IL 6: thermoscientifque, EM2IL6
For the quantification of TNF α: thermoscientifique, EMTNFA
For all tests, the cortex was homogenized after thawing in 50mM Tris-150mM NaCl, pH 7.5 buffer and sonicated for 20 s. After centrifugation (16100g, 15min, 4 ℃), the supernatant or plasma was used for the ELISA assay according to the instructions of the manufacturer of the ELISA assay. At each test, the absorbance was read at 450nm and the concentration of the sample was calculated using a standard curve. Results are expressed in pg marker/mg wet tissue.
All values, except for passive avoidance latency, are expressed as mean ± standard deviation of the measurement. Statistical analysis was performed separately for each compound using one-way analysis of variance (F-number) followed by Dunnett's post-hoc multiple comparison test. Since the upper time limit is fixed, the passive avoidance latency does not follow a gaussian distribution. Therefore, they were analyzed using Kruskal-Wallis nonparametric analysis of variance (H-value) followed by Dunn multiple comparison tests. Values of P <0.05 were considered statistically significant.
The test was performed on 60 male mice, divided into 6 groups of 10, of which group 1 was a negative control group and groups 2-6 were positive control groups:
group 1 was a group to which normal saline was subcutaneously administered instead of D-galactose and kibble a 1.
Group 2 was the group administered D-galactose and kibble a 1;
group 3 was the group administered D-galactose and kibble a 2;
group 4 was the group administered D-galactose and kibble a 3; and
group 5 was the group administered D-galactose and kibble a 4; and
group 6 was the group administered D-galactose and kibble a 5.
The FDA (guidelines, 2005) calculates the definition of the equivalent daily dose for humans from the daily dose tested in mice as follows: the daily human dose in mg/kg body weight (HED humans) is equal to the ratio of the daily animal dose in mg/kg body weight (HED animals) multiplied by the safety factor for the animal in question (Km animals) and the safety factor for humans (Km humans). Km human equals 37 and Km mouse equals 3.
Effect of the Y maze spontaneous alternation test on spatial memory:
results as shown in figure 1, the first panel (left) illustrates the effect of the supplement of the invention on spontaneous alternans disorder, and the second panel (right) illustrates the effect of the supplement of the invention on autonomic activity.
In fig. 1: LOW, LOW dose supplement (a 2); MED, medium dose supplement (a 3); HI, high dose supplement (a 4); n is comprised between 9 and 10, depending on the respective group; p <0.05, # p <0.0001 compared to saline/Veh group, # p <0.05, # p <0.01, # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
It was observed that treatment with D-galactose significantly altered spatial working memory compared to mice treated with physiological saline.
Supplement a2 did not show any effect on the alternation behaviour. Supplement a3 had a rather significant but partially reduced effect on disorders caused by chronic poisoning with D-galactose. Supplement a4 has a rather significant and complete attenuation of disorders caused by chronic poisoning with D-galactose.
Treatment with DHA alone (according to A5) has considerably but partially alleviated the disorders caused by chronic poisoning of D-Gal.
Surprisingly, the results show that the prophylactic treatment with the supplement according to the invention has a considerably more positive effect (considerably and completely attenuating the disorder) than the treatment with DHA alone (considerably and partially attenuating the disorder), with the same dose of DHA.
Effect on D-Gal induced learning disorder according to MWM test:
the results are shown in FIG. 2.
In fig. 2: LOW, LOW dose supplement (a 2); MED, medium dose supplement (a 3); HI, high dose supplement (a 4); n is comprised between 9 and 10, depending on the respective group; p <0.05, p <0.01, # p <0.0001 compared to saline/Veh group, # p <0.01, # p <0.0001 compared to D-GAL 150/Veh group; bonferroni multiple comparison test after two-way anova.
Chronic poisoning by D-galactose has significantly altered spatial learning compared to the negative control group (saline/vehicle).
Supplement a2 did not show any effect on the alternation behaviour.
Supplement a3 had a rather significant but partially reduced effect on disorders caused by chronic poisoning with D-galactose.
Supplement a4 has a rather significant and complete attenuation of disorders caused by chronic poisoning with D-galactose.
According to a5, DHA alone has a rather significant and partial alleviation of disorders caused by chronic poisoning by D-galactose.
Surprisingly, the results show that the prophylactic treatment with the supplement according to the invention at the dose of a4 has a considerably more positive effect (considerably and completely attenuating the disorder) compared to the treatment with DHA alone (considerably and partially attenuating the disorder), at the same DHA dose.
Effect of supplement and DHA on learning impairment caused by D-galactose
The results are shown in FIG. 3.
In fig. 3: LOW, LOW dose supplement (a 2); MED, medium dose supplement (a 3); HI, high dose supplement (a 4); n is comprised between 9 and 10, depending on the respective group; p <0.0001 vs. saline/Veh group,/Veh; # p <0.0001 compared to D-GAL 150/Veh; bonferroni multiple comparison test after two-way anova.
Chronic poisoning by D-galactose has significantly altered spatial learning compared to the negative control group (saline/vehicle).
Supplement a2 did not show any effect on the alternation behaviour.
Supplement a3 had a rather significant but partially reduced effect on disorders caused by chronic poisoning with D-galactose.
Supplement a4 has a rather significant and complete attenuation of disorders caused by chronic poisoning with D-galactose.
According to a5, DHA alone has a rather significant and partial alleviation of disorders caused by chronic poisoning by D-galactose.
Surprisingly, the results show that the prophylactic treatment with the supplement according to the invention at the dose of a4 has a considerably more positive effect (considerably and completely attenuating the disorder) compared to the treatment with DHA alone (considerably and partially attenuating the disorder), at the same DHA dose. Furthermore, the prophylactic treatment with the supplement at the dose of a3 had the same effect (rather significant and partial attenuation of the disorder) as the treatment with DHA alone, whereas the concentration of DHA in the latter was 2 times higher than in the former.
Effect on D-galactose induced Passive avoidance disorder in mice
The results are shown in figure 4, where the effect of the supplement of the invention on the jump latency is shown on the left and the effect on the escape latency is shown on the right, the results being measured during the retention period.
In fig. 4: LOW, LOW dose supplement (a 2); MED, medium dose supplement (a 3); HI, high dose supplement (a 4); n is comprised between 9 and 10, depending on the respective group; p <0.0001 compared to saline/Veh group, # # # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
Chronic poisoning by D-galactose significantly altered long-term situational working memory compared to the negative control group (saline/vehicle).
Supplement a2 did not show any effect on long-term situational working memory.
Supplement a3 was able to attenuate in a non-significant way the disturbances caused by chronic poisoning by D-galactose.
Supplement a4 has a rather significant and complete attenuation of disorders caused by chronic poisoning with D-galactose.
Treatment with DHA alone (according to a5) can attenuate in a non-significant way the disturbances caused by chronic poisoning by D-galactose.
Surprisingly, the results show that the prophylactic treatment with the supplement according to the invention at the dose of a4 has a considerably more positive effect (considerable and complete attenuation of the disorder) compared to the treatment with DHA alone (non-significant attenuation of the disorder), at the same DHA dose. Furthermore, the prophylactic treatment with the supplement at the dose of a3 had the same effect (non-significant attenuation of the disorder) as the treatment with DHA alone, whereas the concentration of DHA in the latter was 2 times higher than in the former.
Effect of supplement and DHA on D-galactose induced lipid peroxidation
The results are shown in FIG. 5.
In fig. 5: LOW, LOW dose supplement (a 2); MED, medium dose supplement (a 3); HI, high dose supplement (a 4); n is comprised between 9 and 10, depending on the respective group; p <0.01, p <0.0001 compared to saline/Veh group, # # # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
Chronic poisoning by D-galactose significantly increased oxidative stress compared to the negative control group (saline/vehicle).
Supplement a2 did not show any effect on lipid peroxidation due to D-galactose chronic poisoning.
Supplement a3 had a rather significant but partially reduced effect on oxidative stress caused by chronic poisoning with D-galactose.
Supplement a4 has a rather significant and complete attenuation of oxidative stress caused by chronic poisoning with D-galactose.
According to a5, DHA treatment alone did not show any effect on oxidative stress caused by chronic poisoning of D-galactose.
Surprisingly, the results show that the prophylactic treatment with the supplement according to the invention at the dose of a4 has a considerably more positive effect (considerably significant and completely reduced oxidative stress) than the treatment with DHA alone, at the same dose.
Effect of supplementation and DHA on D-galactose induced TNF-alpha expression in cortex and plasma
The results are shown in fig. 6, with the left panel showing the effect on the cortex and the right panel showing the effect on plasma.
In fig. 6: LOW, LOW dose supplement (a 2); MED, medium dose supplement (a 3); HI, high dose supplement (a 4); n is comprised between 9 and 10, depending on the respective group; p <0.0001 compared to saline/Veh group, # # # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
Chronic poisoning by D-galactose significantly increased TNF-a in the cortex and plasma compared to the negative control group (saline/vehicle).
Supplements A2 and A3 considerably but in part reduced the increase in TNF- α caused by chronic poisoning with D-galactose in the cortex and plasma.
Supplement a4 reduced TNF-a levels in the cortex and plasma quite significantly and completely.
According to a5, DHA treatment alone had a rather significant but partially reduced effect on the increase of TNF-a caused by chronic poisoning with D-galactose in the cortex and plasma.
Surprisingly, the results show that the prophylactic treatment with the supplement HI (a4) had a considerably more positive effect (considerably and completely reduced increase of TNF- α in the cortex and plasma) than the treatment with DHA alone, at the same dose. Furthermore, prophylactic treatment with the supplements at doses a2 and A3 was as effective (rather significantly reduced) in the cortex and plasma as treatment with DHA alone, with the latter DHA concentrations being 6-fold and 2-fold greater than the prophylactic treatment with the supplements at doses a2 and A3, respectively.
Effect of supplement and DHA on D-galactose induced IL-6 expression in cortex and plasma
The results are shown in fig. 7, with the left panel showing the effect on the cortex and the right panel showing the effect on plasma.
In fig. 7: LOW, LOW dose supplement (a 2); MED, medium dose supplement (a 3); HI, high dose supplement (a 4); n is comprised between 9 and 10, depending on the respective group; p <0.0001 compared to saline/Veh group, # # # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
Chronic poisoning by D-galactose significantly increased IL-6 in the cortex and plasma compared to the negative control group (saline/vehicle).
The low dose supplement (A2) did not show any effect on the concentration of IL-6 caused by D-galactose poisoning.
The medium dose supplement (a3) considerably but partially reduced the increase in IL-6 levels caused by chronic poisoning with D-galactose in the cortex and plasma.
The high dose supplement (a4) considerably and completely reduced the IL-6 content in the cortex and plasma caused by chronic poisoning with D-galactose.
According to a5, DHA treatment alone had a rather significant but partially reduced effect on the increase of IL-6 caused by D-galactose chronic intoxication.
Surprisingly, the results show that the prophylactic treatment with supplement HI of a4 has a considerably more positive effect (considerably and completely reduced increase of IL-6 in the cortex and plasma) than the treatment with DHA alone (considerably and partially reduced increase of IL-6 in the cortex and plasma), at the same dose. In addition, prophylactic treatment with the supplement at the a3 dose had the same impact on the cortex (non-significant attenuation of the disorder) as treatment with DHA alone, with the latter having a DHA concentration 2 times higher than the former.
In summary, the following steps:
chronic poisoning by D-galactose rather significantly causes changes in spatial working memory and long-term situational memory and impairs spatial learning. Behavioral changes are also associated with biochemical changes manifested by an increase in oxidative stress and induction of neuroinflammatory processes.
The preventive treatment of the supplement of the invention is dose-dependent, with a rather marked and completely attenuated effect on the disorders caused by D-galactose chronic intoxication, expressed by behavioral changes, an increase in oxidative stress and activation of neuroinflammatory processes, in the case of the strongest test dose (supplement a 4).
Treatment with DHA alone (a5) had a considerably but partially reduced effect on the disorders caused by D-galactose chronic poisoning, manifested by altered behaviour, increased oxidative stress and activation of neuroinflammatory processes, compared to treatment with supplemental HI at equivalent doses of DHA.
Surprisingly, the prophylactic treatment of the supplement is significantly more effective than the prophylactic treatment with DHA alone at equivalent doses in slowing down the age-related cognitive decline in a mouse model caused by D-galactose chronic poisoning. Furthermore, the prophylactic treatment of the supplement (DHA dose 2 times lower) had the same positive effect as the treatment with DHA alone, in terms of reduction of the oxidative stress measured in the cortex (IL-6 and TNF- α) and in the plasma (TNF- α), in terms of long-term situational memory and in terms of spatial learning, while the DHA concentration of the latter was 2 times higher than that of the former. Moreover, the prophylactic treatment of the supplement had the same positive effect as the treatment with DHA alone, in terms of reducing the oxidative stress measured in the cortex (IL-6 and TNF- α) and in the plasma (TNF- α), the latter having a DHA concentration 6 times that of the former.
Thus, by applying the formula for the calculation of the human equivalent daily dose, the preventive treatment of cognitive decline associated with aging can be defined as the intake of 2-5mg of supplement per kilogram of body weight per day.
Example 3: testing of extracts of the microalgae Isochrysis luteum in young female rats with prenatal stress
In this example, the solution of cognitive deficits, anxiety behavior and alterations in cognitive memory induced by young female rats after prenatal stress in their ancestors by administration of supplements based on extracts of the microalgae dinoflagellates luteum corresponding to the supplements used in example 2 was studied.
Apparatus and method
The model used in this example is a well-established model, and rat prenatal pressure was induced by immobilizing pregnant female rats in cylinders under intense light.
Pregnant female rats were randomly assigned to either prenatal Stress (SP) or control (NS) groups and placed individually in plastic cages with free access to food and water except during behavioral testing. The conditions in the cage were as follows: the light cycle was 12h light/12 h dark cycle (light on 7 am), in a constant temperature (21 ℃) and constant humidity (50%) room.
The prenatal pressure procedure was performed as described by Meuner et al (2004). Fixation of female rats is the subject of a semi-random confinement procedure. The animals were placed and kept in transparent ferret fixators (ferret retainers) made of plexiglass (length 20cm, diameter 7cm) under intense light for a total of 90 minutes per day for 4 consecutive days. In order to make the pressure as unpredictable as possible, a forced immobilization of 90 minutes was carried out in the following manner: a single 90 minute phase, two 45 minute phases 4 hours apart, two 60 and 30 minute phases 4 hours apart, or three 30 minute phases 4 hours and 1 hour apart, and at different times of the day.
The control group of dams were also manipulated, but never placed in ferret holders.
On postnatal day 1 (PPD1), the treated female rats were allowed to spontaneously escape the restraint of the ferret immobilizer.
The pups had been weaned at PPD 21. Rats were separated from the mother rats, identified according to their sex, weighed, and rats of the same sex were assigned to cages (3 rats per cage). Pups in the same cage were from different litters to avoid any possible litter-related effects.
Conditions within the cage were as follows: the light cycle was 12h light/12 h dark cycle (light on 7 am), and food and water were freely available in a constant temperature (21 ℃) and constant humidity (50%) room, except for the behavioral testing time.
In each cage, animals received the same treatment. These animals were tested randomly and in a double-blind manner.
48 female rats were used and divided into four groups of animals, which were constructed in the following manner:
group 1 consisted of 12 control female rats, i.e., their ancestors were not subjected to prenatal stress, and received only 200. mu.L of vehicle solution (labeled: NS/vehicle) per day. Thus, this group is a control group;
group 2 consisted of 12 control female rats, i.e. their ancestors were not under prenatal stress and received 200 μ L of supplement (labeled: NS/supplement) daily.
Group 3 consisted of 12 female rats, whose ancestors had been subjected to prenatal stress, receiving 200 μ L of carrier solution (labeled: SP/carrier) per day.
Group 4 consisted of 12 female rats, whose ancestors had been subjected to prenatal stress, receiving 200 μ L of supplement per day (labeled: SP/supplement).
The effectiveness of supplements has been evaluated 6 weeks after birth.
The supplement (one dose) was gavaged once a day for 5 days per week. Administration was initiated post weaning, i.e. after the 25 th day of postpartum (PPD), and continued until PPD 46.
The daily intake was 25.7 mg of supplement per kg of rat body weight.
Animals were behavioral tested between days PPD46 and PPD48, i.e., outside the period of treatment with the vehicle or supplement. Thus, the effect observed in the behavioral test will be due to the treatment being prophylactic in nature.
The behavior test is divided into an anxiety assessment link and two object identification links. Links are defined as follows:
link 1, PPD 46: rats were individually placed in a square open space (50cm x 50cm) made of blue plexiglass, and the floor was fitted with an infrared sensorA light emitting diode. Rats were habituated to the test space for 10 minutes, and their displacement was captured by an infrared camera and used
Figure BDA0003221927560000221
(Noldus) software. Activity was analyzed in terms of distance (m) covered overall and percentage of occurrences within the software defined central area of 25cm x 25cm, and these data report the intensity of anxiety behavior (38).
Link 2, PPD 47: two identical objects (50mL plastic Eppendorf tubes) were placed at defined positions (at the two opposite edges of the central area). Each rat was placed in the test space and the exploratory activity was recorded over a period of 10 minutes. The activity is analyzed in terms of the number of contacts with the object and the duration of the contacts.
Link 3, PPD 48: the object of link 2 is replaced with a new object (a plastic bottle cap) having a shape, texture and color different from the familiar object. Each rat was returned to the test space and the exploration activity was recorded over a period of 10 minutes. The analysis of this activity is similar to that described in link 2.
The calculation method of the priority exploration index comprises the following steps: the ratio of the number of contacts (or duration) with the object of link 2 to the total number of contacts (or duration) with both objects.
All values are expressed as mean ± standard deviation of the measurement. Statistical analysis was performed separately for each compound using one-way analysis of variance (F-number) followed by Dunnett's post-hoc multiple comparison test.
The FDA (guidelines, 2005) calculates the definition of the equivalent daily dose for humans from the daily dose tested in rats as follows: the daily human dose in mg/kg body weight (HED humans) is equal to the ratio of the daily animal dose in mg/kg body weight (HED animals) multiplied by the safety factor for the animal in question (Km animals) and the safety factor for humans (Km humans). Km human equals 37 and Km rat equals 6.
The results are shown below.
Test spatial center movement, day PPD 46; supplementEffect of Agents on anxiety
The results are shown in FIG. 8.
In fig. 8: effects of treatment on anxiety. N-12; p <0.0001 relative to treatment group NS/vector; relative to treatment group SP/vehicle, ### # p < 0.0001; dunnett test.
The SP/vector group corresponds to individuals who were subjected to prenatal stress and were prophylactically treated with vector alone, and the percentage of displacement in the peripheral region of the open test space was significantly higher than that of the NS/vector group (group not subjected to prenatal stress).
The SP/supplement group corresponds to individuals who suffered prenatal stress and were prophylactically treated with supplements, with a percentage of displacement in the peripheral area of the open test space significantly lower than the SP/carrier group (group who suffered prenatal stress but did not receive supplement treatment). In addition, the% shift for the SP/supplement group was equal to the% shift for the control NS/vector.
The displacement rate of individuals in the peripheral region of the open test space is higher than that of the control pattern, which is an expression of anxiety behavior [63], and the uncovered area is restricted and monitored by a protection mechanism based on the search boundary.
Thus, Prenatal Stress (PS) has caused very significant anxiety behavior.
Surprisingly, the results show that the supplement has considerably reduced the anxiety behaviour caused by prenatal stress.
Identification test, day PPD 47; effect of supplements on cognitive memory in object recognition
The results are shown in FIG. 9.
In this link, the same object is presented to the individual twice.
For this parameter, no statistical effect between groups was measured.
Thus, all population individuals interact in the same way when they are in contact with the same object, their interactions being evenly distributed (50%) between the two objects both in frequency and duration.
Identification test, day PPD48 (new object);effect of supplements on recognition memory of tests for recognizing new objects
The results are shown in FIG. 10.
In fig. 10: n-12; p <0.0001 relative to treatment group NS/vector; relative to treatment group SP/vehicle, ### # p < 0.0001; dunnett test.
In this link, two different objects are presented to the individual once: one of the objects corresponds to the object displayed in link 2 and the other is a new object.
The SP/vector group corresponds to individuals who were subjected to prenatal stress and were prophylactically treated with vector alone, and the percentage of interaction with the presented new subjects was significantly lower in terms of frequency and duration than the NS/vector group (group not subjected to prenatal stress). And this percentage is equal to the percentage obtained in link 2 for all groups. Thus, individuals of the SP/carrier group interact as many with old objects as with new objects, and therefore the individuals of the group cannot identify the old objects displayed in link 2.
In contrast, the SP/supplement group corresponds to individuals who suffered prenatal stress and received prophylactic treatment with supplements, whose percentage of interaction with the presented new subjects is significantly higher in terms of frequency and duration than the PS/vehicle group (negative control group). And this percentage is higher than the percentage obtained in link 2 for all groups. Thus, the SP/supplement group individuals interacted less with the old than with the new, and thus the group individuals identified the old displayed in link 2. Furthermore, the percentage of interaction of the individuals of the SP/supplement group with the displayed new object, whether in frequency or time, was equal to that of the individuals of the NS/carrier group and NS/supplement group.
Thus, the prenatal Pressure (PS) causes a very significant recognition memory impairment in the case of new objects.
Surprisingly, the results show that the supplement is able to considerably and completely attenuate the cognitive memory impairment caused by prenatal stress.
In summary, the following steps:
treatment with the supplement significantly and completely attenuates anxiety behavior caused by prenatal stress and cognitive memory impairment.
The prenatal stress in this experiment caused anxiety behavior in young female rats significantly and altered cognitive memory quite significantly.
Thus, by applying the calculation of the human equivalent daily dose, a prophylactic treatment for reducing prenatal stress induced cognitive disorders can be defined as the intake of 0.05-0.1mg of supplement per kg of body weight per day.
Example 4: natural extract of microalgae phaeodactylum tricornutum in the context of in vivo models reduced phase of aging Testing of disorders caused by cognitive decline of concern
The food supplement of the present invention is prepared from Phaeodactylum tricornutum extract comprising (mg/g):
omega-3 type fatty acids (ALA, SDA, EPA, DHA): 66.6 plus or minus 11.5;
fucoxanthin: 20.0 plus or minus 4.0;
sterol: 3.0 plus or minus 0.6;
algae prostacyclin: 0.0025 + -0.0005.
The supplement is obtained by adding 410mg + -20mg/g of coconut oil to the extract.
The supplements were added to the kibbles according to 4 different formulas, the amounts of supplements added in different batches of kibbles were in agreement with the human equivalent daily dose described in table 2, and the final quality of all formulas was the same by diluting the compositions described below in coconut oil.
The FDA (guidelines, 2005) calculates the definition of the equivalent daily dose for humans from the daily dose tested in mice as follows: the daily human dose in mg/kg body weight (HED humans) is equal to the ratio of the daily animal dose in mg/kg body weight (HED animals) multiplied by the safety factor for the animal in question (Km animals) and the safety factor for humans (Km humans). Km human equals 37 and Km mouse equals 3.
Another batch of kibbles was formulated with only coconut oil, so that the carrier concentration was equal to the other batches, i.e., 0.01% (m: m).
The five batches of kibble thus obtained are described in table 2 below.
[ Table 2]
Figure BDA0003221927560000261
The in vivo model considered is a D-galactose model applied to mice, which is suitable for studying cognitive decline associated with aging. In fact, this model mimics many of the behavioral and molecular characteristics of brain aging in rodent models.
D-galactose was administered subcutaneously at a rate of 150mg/kg mouse wet weight per day and the food supplement described herein was incorporated into the pellets, following the following pattern:
between day-28 and day 51, the supplement is administered by incorporation into a food pill.
-subcutaneously administering D-galactose 5 days weekly from day 01 to day 51.
Between day 43 and day 51, three different behavioral tests were used to monitor the effect of the test compounds.
The effectiveness of the supplement was evaluated according to the following parameters: improvement of learning disabilities (spatial working memory: spontaneous alternation according to the Y maze test; spatial memory by the so-called "Morris water maze" and long-term situational memory by passive avoidance test), Lipid Peroxidation (LPO) rate of hippocampus and effect on neuroinflammatory markers IL6 and TNF α.
Amelioration of learning disabilities
On day 43, all animals tested spontaneous alternation in the Y Maze (YM) test by spatial working memory index.
All animals were tested for spatial memory in the Morris Water Maze (MWM) test by spatial memory index from day 44 to day 49.
All animals passed the MWM test to assess spatial working memory from day 44 to day 49.
-assessing the long-term situational memory of the animal with a stepwise passive avoidance procedure (STPA) on days 50 and 51, respectively, by exercise and retention sessions.
Lipid Peroxidation (LPO) rate of hippocampus and effects on neuroinflammatory markers IL6 and TNF α
On day 51, animals were euthanized at the end of the behavioral testing.
For all animals, trunk blood was collected and centrifuged to recover plasma, and brains were collected rapidly. Dissecting hippocampus and cortex, and determining lipid peroxidation rate by colorimetry using hippocampus; the prefrontal cortex and plasma are used to determine the levels of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha).
Quantification of Lipid Peroxidation (LPO) rates was performed according to the Hermes-Lima et al modified and adapted procedure. The method measures the ability of brain lipid peroxides to oxidize the ferrous oxide and xylenol orange complex, performed in the presence of cumene Hydroperoxide (HPC). Lipid peroxidation levels were determined as HPC equivalents according to the following formula:
HPCE＝A5801/A5802 x[HPC(nmol)]。
and expressed as HPC equivalents per wet tissue weight and as a percentage of the data obtained with the control (D-galactose + vehicle).
The levels of IL6 and TNF α were quantified by ELISA assay using the following kit:
for quantification of IL 6: thermoscientifque, EM2IL6
For the quantification of TNF α: thermoscientifique, EMTNFA
For all tests, the cortex was homogenized after thawing in 50mM Tris-150mM NaCl, pH 7.5 buffer and sonicated for 20 s. After centrifugation (16100g, 15min, 4 ℃), the supernatant or plasma was used for the ELISA assay according to the instructions of the manufacturer of the ELISA assay. At each test, the absorbance was read at 450nm and the concentration of the sample was calculated using a standard curve. Results are expressed in pg marker/mg wet tissue.
All values, except for passive avoidance latency, are expressed as mean ± standard deviation of the measurement. Statistical analysis was performed separately for each compound using one-way analysis of variance (F-number) followed by Dunnett's post-hoc multiple comparison test. Since the upper time limit is fixed, the passive avoidance latency does not follow a gaussian distribution. Therefore, they were analyzed using Kruskal-Wallis nonparametric analysis of variance (H-value) followed by Dunn multiple comparison tests. Values of P <0.05 were considered statistically significant.
The test was performed on 60 male mice, divided into 6 groups of 10, of which group 1 was a negative control group and groups 2-6 were positive control groups:
group 1 was a group to which normal saline was subcutaneously administered instead of D-galactose and kibble B1.
Group 2 was the group administered D-galactose and kibble B1;
group 3 was the group administered D-galactose and kibble B2;
group 4 was the group administered D-galactose and kibble B3; and
group 5 was the group administered D-galactose and kibble B4; and
group 6 was the group administered D-galactose and kibble B5.
Effect of the Y maze spontaneous alternation test on spatial memory:
results as shown in fig. 11, the first graph (left) illustrates the effect of the supplement of the present invention on spontaneous alternation disorder, and the second graph (right) illustrates the effect of the supplement of the present invention on autonomic activity.
In fig. 11: normal saline/vehicle corresponds to negative control (group fed with kibble formulated without D-galactose nor vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; p <0.05, # p <0.0001 compared to saline/Veh group, # p <0.05, # p <0.01, # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
It was observed that treatment with D-galactose significantly altered spatial working memory compared to mice treated with physiological saline.
The supplement D1 had a rather significant but partially reduced effect on the disorders caused by chronic poisoning with D-galactose. Supplements D2, D3 and D4 had a rather significant and complete attenuation of the disorders caused by chronic poisoning with D-galactose.
Treatment with DHA alone (according to A5) has considerably but partially alleviated the disorders caused by chronic poisoning of D-Gal.
Effect on D-Gal induced learning disorder according to MWM test:
the results are shown in FIG. 12.
In fig. 12: normal saline/vehicle corresponds to negative control (group fed with kibble formulated without D-galactose nor vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; p <0.05, p <0.01, # p <0.0001 compared to saline/Veh group, # p <0.01, # p <0.0001 compared to D-GAL 150/Veh group; bonferroni multiple comparison test after two-way anova.
Chronic poisoning by D-galactose has significantly altered spatial learning compared to the negative control group (saline/vehicle).
The supplement D1 had a rather significant but partially reduced effect on the disorders caused by chronic poisoning with D-galactose.
Supplements D2, D3 and D4 had a rather significant and complete attenuation of the disorders caused by chronic poisoning with D-galactose.
Effect of supplements on learning disorders caused by D-galactose
The results are shown in FIG. 13.
In fig. 13: normal saline/vehicle corresponds to negative control (group fed with kibble formulated without D-galactose nor vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; normal saline/vehicle corresponds to negative control (group fed with kibble formulated without D-galactose nor vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; p <0.0001 vs. saline/Veh group,/Veh; # p <0.0001 compared to D-GAL 150/Veh; bonferroni multiple comparison test after two-way anova. "T", the time spent in the target quadrant; "O", the average time spent in the other three quadrants.
Chronic poisoning by D-galactose has significantly altered spatial learning compared to the negative control group (saline/vehicle).
Supplements D1 and D2 had a rather pronounced but partially attenuated effect on disorders caused by chronic poisoning with D-galactose.
Supplements D3 and D4 had a rather significant and complete attenuation of disorders caused by chronic poisoning with D-galactose.
Effect on D-galactose induced Passive avoidance disorder in mice
The results are shown in figure 14, where the effect of the supplement of the invention on the jump latency is shown on the left and the effect on the escape latency is shown on the right, measured over the retention period.
In fig. 14: normal saline/vehicle corresponds to negative control (group fed with kibble formulated without D-galactose nor vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; p <0.0001 compared to saline/Veh group, # # # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
D-galactose significantly altered long-term situational working memory compared to the negative control group (saline/vehicle).
Supplement D1 did not show any effect on long-term situational working memory.
Supplements D2, D3 and D4 had a rather significant and complete attenuation of the disorders caused by chronic poisoning with D-galactose.
Effect of supplements on D-galactose induced lipid peroxidation
The results are shown in FIG. 15.
In fig. 5: normal saline/vehicle corresponds to negative control (group fed with kibble formulated without D-galactose nor vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; p <0.01, # p <0.0001 compared to saline/Veh group, # p <0.01, # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
Chronic poisoning by D-galactose significantly increased oxidative stress compared to the negative control group (saline/vehicle).
The supplement D1 had a rather significant but partially reduced effect on oxidative stress caused by chronic poisoning with D-galactose.
Supplements D2, D3 and D4 have a rather significant and complete attenuation of oxidative stress caused by chronic poisoning with D-galactose.
Effect of supplements on D-galactose induced TNF-alpha expression in cortex and plasma
The results are shown in fig. 16, with the left panel showing the effect on the cortex and the right panel showing the effect on plasma.
In fig. 16: normal saline/vehicle corresponds to negative control (group fed with kibble not treated with D-galactose and formulated with vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; p <0.0001 compared to saline/Veh group, # # # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
Chronic poisoning by D-galactose significantly increased TNF-a in the cortex and plasma compared to the negative control group (saline/vehicle).
Supplement D1 reduced considerably but in part the increase in TNF-a caused by chronic poisoning with D-galactose in the brain and plasma.
Supplement D2 reduced the increase in TNF-a in the brain due to chronic poisoning with D-galactose quite significantly and completely, but partially in the plasma.
Supplement D3 reduced the increase in TNF-a in the brain due to chronic poisoning with D-galactose quite significantly but in part, but completely in the plasma.
Supplement D4 reduced considerably and completely the increase in TNF-a caused by chronic poisoning with D-galactose in brain and plasma.
Effect of supplements on D-galactose induced IL-6 expression in cortex and plasma
The results are shown in fig. 17, with the left panel showing the effect on the cortex and the right panel showing the effect on plasma.
In fig. 17: normal saline/vehicle corresponds to negative control (group fed with kibble formulated without D-galactose nor vehicle, coconut oil); DGal 150/Veh corresponds to a positive control (group fed with kibbles treated with D-galactose and formulated with vehicle, coconut oil); d1, D2, D3 and D4 increase the dose of the supplement; n is comprised between 11 and 12, depending on the respective group; p <0.0001 compared to saline/Veh group, # # # p <0.0001 compared to D-GAL 150/Veh group; dunnett test.
Chronic poisoning by D-galactose significantly increased IL-6 in the cortex and plasma compared to the negative control group (saline/vehicle).
Supplements D1 and D2 considerably but in part reduced the increase in IL-6 levels caused by chronic poisoning with D-galactose in brain and plasma.
Supplements D3 and D4 reduced the IL-6 content in brain and plasma due to chronic poisoning by D-galactose quite significantly and completely.
In summary, the following steps:
chronic poisoning by D-galactose rather significantly causes changes in spatial working memory and long-term situational memory and impairs spatial learning. Behavioral changes are also associated with biochemical changes manifested by an increase in oxidative stress and induction of neuroinflammatory processes.
The preventive treatment of the supplement of the invention is dose-dependent, with a rather marked and completely attenuated effect on the disorders caused by chronic poisoning with D-galactose, expressed as a change in behaviour, an increase in oxidative stress and activation of neuroinflammatory processes, in the case of the strongest tested dose (supplement D4). Whereas at medium low doses (supplements D2 and D3), the supplement of the invention has a rather marked but partially attenuated effect on the disorders caused by D-galactose chronic poisoning, which are manifested by spatial working memory, increased oxidative stress and activation of neuroinflammatory processes.
Thus, by applying the formula for the calculation of the equivalent daily dose in humans, the prevention of age-related cognitive decline can be defined as the intake of 1.7-5.3mg supplement/kg body weight per day.

Claims (18)

1. A composition comprising at least 50mg/g of one or more omega-3 type fatty acids, at least 10mg/g of one or more xanthophylls, at least 1mg/g of one or more sterols, and at least 2 μ g/g of one or more algal prostaglandins (phycopostanes).
2. The composition of claim 1, wherein the composition comprises 50-250mg/g of one or more omega-3 fatty acids, 10-50mg/g of one or more xanthophylls, 1-20mg/g of one or more sterols, and 2-100 μ g/g of one or more algal prost.
3. The composition of claim 1 or 2, wherein the composition comprises 50-200mg/g of one or more omega-3 type fatty acids, 10-30mg/g of one or more xanthophylls, 1-8mg/g of one or more sterols, and 2-50 μ g/g of one or more algal prostins.
4. The composition of any one of claims 1 to 3, wherein the composition comprises 50 to 170mg/g of one or more omega-3 type fatty acids, 10 to 25mg/g of one or more xanthophylls, 1 to 6mg/g of one or more sterols, and 2 to 40 μ g/g of one or more algal prostins.
5. A food supplement comprising the composition of any one of claims 1-4 and at least one oil selected from Medium Chain Triglycerides (MCT).
6. The food supplement according to claim 5, wherein the Medium Chain Triglycerides (MCT) are selected from coconut oil and palm oil.
7. The composition according to any one of claims 1 to 4 or the food supplement according to claim 5 or 6, characterized in that the omega-3 type fatty acids or at least one omega-3 type fatty acid is selected from stearic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and mixtures thereof.
8. The composition according to any one of claims 1 to 4 and 7 or the food supplement according to any one of claims 5 to 7, characterized in that the lutein or at least one lutein is fucoxanthin.
9. The composition according to any one of claims 1 to 4, 7 and 8 or the food supplement according to any one of claims 5 to 8, wherein the or at least one sterol is selected from phytosterols.
10. The composition according to any one of claims 1 to 4 and 7 to 9 or the food supplement according to any one of claims 5 to 9, characterized in that the algal prost or at least one algal prost is selected from the group consisting of vegetal prost, isoprost and neuroprost.
11. The composition according to any one of claims 1 to 4 and 7 to 10 or the food supplement according to any one of claims 5 to 10, further comprising at least one additive selected from preservatives, colouring agents, flavouring agents, disintegrating agents, lubricants, coating agents or encapsulating agents.
12. Composition according to any one of claims 1 to 4 and 7 to 11 or food supplement according to any one of claims 5 to 11, characterized in that it is in the form of a gel capsule, tablet, lozenge or loose powder.
13. Composition according to any one of claims 1-4 and 7-12 or food supplement according to any one of claims 5-12, characterized in that it is packaged in doses having a unit weight between 10mg and 1 g.
14. Use of microalgae selected from any one of the classes lipophyceae, chrysophyceae, diatoms, phakophyceae, proliniophyceae, dinoflagellates, chrysophyceae, isodinoflagellates and phaeodactylaceae for the preparation of a composition according to any one of claims 1-4 and 7-13 or a food supplement according to any one of claims 5-13.
15. The use according to claim 14, wherein the microalgae is dinoflagellate luteum or phaeodactylum tricornutum.
16. Use of the food supplement according to any of claims 5-13 for the prevention of the appearance of age-related cognitive disorders defined as non-pathological cognitive decline, or for the prevention of cognitive disorders in young adults or children suffering from prenatal stress that triggers non-pathological cognitive disorders such as hyperactivity, attention and memory deficits, language retardation and anxiety behavior.
17. Use of a food supplement according to claim 16 for the prevention of the appearance of cognitive disorders associated with aging, defined as non-pathological decline of cognitive function, characterized by the ingestion of 2-5mg extract per kg body weight per day.
18. Use of the food supplement according to claim 16 for the prevention of cognitive disorders in young adults or children suffering from prenatal stress, which causes non-pathological disorders such as hyperactivity, attention and memory deficits, delayed language development and anxiety behaviour, characterized by the daily intake of 0.05-0.1mg extract per kg body weight.
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